Temperature-Dependent Development of the Swallowtail Butterfly, Sericinus montela Gray

Abstract

Seong-Jin Hong et al. Characteristics of the swallowtail butterfly 154 155 sequestration in the body (Omura et al., 2006). For instance, larvae secrete odoriferous fluid from their defensive glands (osmeteria), which contain five of the eight aristolochic acid compounds found in the host, A. debilis (Nishida, 1995). S. montela is dimorphic at the adult stage, which is characteristic of primitive insects. In the wild, it is simple to distinguish males and females by the color of their wings. Male wings are bright beige, whereas females have dark brown wings that are described as a melanized pattern (Nam et al., 1998), which has not yet been proven. Like most other butterflies, S. montela adults are solitary in behavior. Females lay clutches containing dozens to a hundred eggs on the stems or leaves of the host plant (Shin, 1974). After 5–8 d, larvae hatch from the eggs in a group and pass through five instars with a total developmental time of 16–21 d, depending on the ambient thermal environment and availability of host plants in Korea. Adults survive for about two weeks after pupae metamorphogenesis, which takes 10–12 d unless they enter diapause for overwintering. Taken together, adult emergence takes approximately one month, and completion of the full life cycle lasts up to about 50 d in the field. However, Russian subspecies take 16–19 d for the adult to develop at 20–25°C in the laboratory (Monastyrskiy and Kotlobay, 1995). Therefore, S. montela is multivoltine, emerging two or three times a year on average in Korea (Shin, 1974; Kim and Lee, 1992; Nam et al., 1998). The habitat of S. montela in the Gwangneung area, one of the most important sites for insect conservation in Korea, was investigated for a long period of time from 1932 to 2004 (Shin, 1974; Byun et al., 2005). Swallowtail emergence was observed three times a year from 1960 to 1962 (Shin, 1974). Since then, the population has decreased rapidly until only a single male was observed in April 2004, despite the presence of 148 species (67.0% of the S. Korea butterfly fauna) listed in Gwangneung Forest (Byun et al., 2005). As a part of the ecosystem, the world’s human population has continuously increased, and it predominantly occupies most of the earth while simultaneously destroying or fragmenting natural habitats, which threatens worldwide biodiversity (Fahrig, 2003; Kim, 2010). Global warming caused by the greenhouse effect is a pernicious threat to swallowtails (Collins and Morris, 1985; Collins, 1991), and many species, including S. montela, are facing environmental challenges or further threats. Particularly, because S. montela habitats are intimately associated with human-occupied areas, the species is exposed to additional variety of butterfly specimens, especially swallowtails, have been on display all over the world, and live butterflies are exhibited in indoor gardens and museums for educational and aesthetic purposes. A representative flight mode of butterflies is fluttering, which is conspicuous because of large flamboyant wings. In addition, the wing couple is attached to the second and third thoraces, but it is not hooked together. This characteristic and the lack of bristles allow their flight to be more elegant. Possible correlations between flight performance and wing shape in swallowtails have been suggested, which might explain the wide range of complex flight patterns (Betts and Wootton, 1988). Field observations of S. montela have described fluttering with slow, full strokes when flying upward or climbing (Shin, 1974; Kim and Lee, 1992). However, the downward flight and landing mode utilize gliding without fluttering. S. montela spreads its wings when basking or landing instead of folding them vertically (Nam et al., 1998). Therefore, the distinct flight of S. montela adds celestial imagery to the beauty of the wings, and this is likely due to its large forewings and particularly long hindwing tails. Approximately 20,000 butterfly species belong to a small part of the insect order Lepidoptera, which also contains the moth group that is ten times larger (Collins, 1991). Butterflies are divided into five families that are predominantly found in the tropics, but a few species extend to cool temperature latitudes in both hemispheres (Collins, 1991). Most swallowtails are distributed in extremely limited areas rather than widespread areas that are sensitive to habitat and environmental changes. S. montela is one of the over 500 species in Papilionidae (Munroe and Ehrlich, 1960), which is endemic to some countries of northeastern Asia, including Russia, China, and Korea (Ackery, 1975; Collins and Morris, 1985). Geographical forms from the northern region (e.g., ssp. greyi Fixs.) as well as the central and southern regions (e.g., ssp. koreana Fixs.) have been assigned to the subspecies found in Korea (Kim and Hong, 1991). However, extensive research on this species has been published mostly in Japan following introductions from Korea as recent as the late 1970s (Kim and Hong, 1991; Kim and Lee, 1992; Kumon, 1996). S. montela feeds exclusively on Aristolochia contorta Bunge (Aristolochiaceae), which is a source of aristolochic acids in plants (Mix et al., 1983). Aristolochic acids, which can be toxic to humans via inhalation or contact with skin (Merk index), protect S. montela caterpillars and adults from predators via Int. J. Indust. Entomol. Vol. 29, No. (2), pp. 153-161 (2014) 154 155 investigation in the Division of Applied Entomology at the National Academy of Agricultural Science. They were grown at 25 ± 1°C and 50 ± 10% relative humidity with a photo regime of 16 L: 8 D, and experiments were conducted after two generations to minimize maternal effects. All S. montela adults and larvae fed on A. contorta plants. The larvae were kept under the following conditions: a small Petri dish (35 mm in diameter and 10 mm in height; 35 mm × 10 mm) for the first stage; a large Petri dish (60 mm × 10 mm height) for the second and third stages; and a large Petri dish (100 mm × 40 mm height) for the final stage. Experimental protocol Female butterflies were placed individually in a box, and were hand-mated in order to obtain eggs (Platt, 1969). Each male was removed from the box after mating, allowing the female to lay eggs on A. contorta. The female was then released from the cage, and the number of eggs adhering to the stem was counted. The eggs were distributed into chambers set at nine temperature regimes (15.0, 17.5, 20.0, 22.5, 25.0, 27.5 30.0, 32.0, and 35°C) with 50 ± 10% relative humidity and a 16 L: 8 D photoperiod. Each temperature-specific chamber contained 30 eggs in triplicate. Survivorship and developmental durations from eggs to larvae and larvae to pupae were recorded on a daily basis. On the initial experimental day, the newly hatched larvae grown at 25.0°C were distributed into various temperatures (15.0, 20.0, 25.0, 30.0 and 35.0°C). The food was replenished regularly each morning after examining larval development. When the fifth instar larvae stopped eating, each individual was placed into a separate large Petri dish (100 mm × 40 mm) where they were allowed to pupate. In addition, when larvae that were consistently reared at 25.0°C reached a pupal stage, they were distributed into 15.0, 20.0, 25.0, 30.0, and 35.0°C temperature regimes to investigate pupal development and adult eclosion. The duration of the each stadium (until pupation) as well as the survival rate was recorded to the nearest day. Statistical analysis Differences in the developmental period of egg, larval, and adult stages were tested by analysis of variance (ANOVA). If significant differences were detected, multiple comparisons were made using Tukey's HSD multiple range test (p = 0.05). dangers. Therefore, the butterfly has disappeared near hiking trails, fields, and stream banks where it was previously observed (Kim, 2010). Special attention should be paid to the development of strategies to protect them (Collins and Morris, 1985; Collins, 1991). Since 2005, Hyundai Motor Company has made efforts to reconstitute natural environments that are optimal for S. montela by establishing eight ecology laboratories (the so-called “The Ecology Learning Center”) in five cities as one of the social responsibilities of the company (http://www.hyundai-green.com). Physiological and ecological research on S. montela should not only be conducted to provide butterfly habitats and guidelines for introducing them into the field, but it should also support the maintenance of optimal habitats and population control in the ecosystem. In insects, their physiological processes, behaviors, and survival are susceptible to environmental variations. Temperature, which is one of the parameters, significantly affects gene expression, body size, and color during development, and it can also affect the natural habitats and generation times of species (Blau, 1981; Smith, 1991; Nylin and Gotthard, 1998; Hazel, 2002; Pateman et al., 2012; Kvist et al., 2013). For that reason, a long-standing issue in biology has been the impact of ambient temperature on physiological processes in the biosystem. As many studies of insects have documented, the growth rate increases with increasing temperature to a maximum point, after which increases in temperature result in larval death (Howe, 1967; Guppy, 1969; Poston et al., 1977; Rawlins and Lederhouse, 1981). In the beginning of the S. montela physiological study, we set out to investigate its developmental characteristics and the effects of ambient thermal environments on embryonic and larval development throughout all developmental stages. Our study focuses on an examination of developmental durations and survival rates of eggs, larvae, and pupae under varying temperatures, with the expectation of discovering the most favorable conditions for laboratory rearing. Materials and Methods Experimental insects Native S. montela individuals were collected